Gal(bl-3)-GalNAc bearing glycoconjugates are concentrated to the axolemmal region of nodes of Ranvier in mature myelinated nerve fibers; a region which contains high levels of voltage sensitive sodium channels. This observation raises the possibility that they may have an obligatory colocalization with sodium channels and the epitopes may be expressed on the channel itself. Peanut agglutinin (PNA) binds to Gal(b1-3)-GalNAc epitopes and cholera toxin binds to GM1-like epitopes. In order to determine whether these moieties could be spatially dissociated on the axolemma from sodium channels, fluorescent and peroxidase labeled PNA and cholera toxin along with antibodies against voltage sensitive sodium channels were used to stain amyelinated axons from the spinal roots of the dystrophic mouse (129 ReJ Dy +/+). Spinal roots from these mice contain many large diameter axons which carry discrete clusters of sodium channels on the axolemma without intervening Schwann cells. In mye linated fibers from normal mice, PNA and cholera toxin binding co-localized with sodium channel immunoreactivity at nodes of Ranvier. Cholera toxin also stained the paranodal region of Schwann cells. In amyelinated axons from the spinal roots of dystrophic mice, PNA and cholera toxin binding occurred mainly in a diffusion and continuous manner on the axolemma. Some patches of cholera toxin binding were observed on the axolemma, but double labeling experiments revealed that these patches did not co-localize with sodium channel clusters. Cholorform-methanol extraction of normal nerve indicated that PNA binds to Gal(b1-3)-GalNAc bearing glycoproteins and cholera toxin to the ganglioside GM1. These results suggest that sodium channel and Gal(b1-3)-GalNAc bearing glycoconjugates do not have an obligatory co-localization with sodium channels and that Gal(b1-3)-GalNAc is most likely a component of gangliosides and glycoproteins associated with the axolemma.